Project Summary/Abstract: Natural product glycosylation is vital for the discovery of new pharmaceutical compounds. While it has been recognized for some time that many biologically active natural products owe much of their therapeutic properties to their glycosylation diversity, methods for creating further diversity for drug discovery have been difficult to develop. 1 of the challenges is that the modified and rare sugars needed for creating diversity are unavailable or extremely difficult to make, and that the sugars need to be chemically "activated" in order to transfer them to the core natural product molecule. However, a new technology, glycorandomization, has been developed, which is a chemoenzymatic method of attaching sugars to natural production aglycons. With this method, a combination of a kinase and nucleotide transferase is needed to form the specific activated sugar and finally a glycosyltransferase is used to transfer the sugar to an appropriate aglycon core. This technology is valuable for drug discovery, because it can be used to produce a library of natural product molecules that are differentially glycosylated. Our proposal is intended to continue the development of this technology by creating a library of activated sugars to feed into it. Specifically, we intend to 1) expand the substrate range of the kinase GalK on selected L-sugars using directed evolution, 2) couple the kinase and transferase reaction, and 3) demonstrate the gram-scale synthesis of an activated sugar. The overall goal of this proposal is to create a robust 1 pot, in vitro, multi-gram scale synthesis of activated sugars for drug discovery and biochemical research purposes. Project Narrative: This project is aimed toward developing a method for making a variety of activated sugars for drug discovery purposes. These sugars can be used to make derivatives of natural products with new therapeutic properties, for example, antibiotics that are effective against antibiotic-resistant bacteria. [unreadable] [unreadable] [unreadable]